Characterization of Microsatellite Loci in Red Wood Ants Formica (S. Str.) Spp

Molecular Ecology Notes (2004) 4, 200–203 doi: 10.1111/j.1471-8286.2004.00614.x

PRIMERBlackwell Publishing, Ltd. NOTE Characterization of microsatellite loci in red wood ants Formica (s. str.) spp. and the related genus Polyergus

EISUKE HASEGAWA* and SHUICHIRO IMAI† *Laboratory of Animal Ecology, Department of Ecology and Systematics, Graduate School of Agriculture and †Laboratory of Animal Ecology, Department of Ecology and Systematics, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

Abstract Ants are interesting subjects for studies of evolution of altruism. We developed 13 microsatellite loci in a red wood ant Formica (s. str.) yessensis from random amplified polymorphic DNA fragments to study genetic structure within populations and colonies. Five loci bore two to five alleles in both F. (s. str.) yessensis and F. (s. str.) truncorum and two were also polymorphic in a related species, Polyergus samurai. Results suggest that the loci will be useful in evolutionary studies on Formica and Polyergus species. Keywords: ants, Formica truncorum, Formica yessensis, microsatellite loci, Polyergus Received 30 August 2003; revision received 24 September 2003; accepted 13 January 2004

The evolution of polygyny is a major issue in evolutionary Thus, new microsatellite loci are useful in studies on biology because this phenomenon can be regarded as the Japanese red wood ants. ‘2nd evolution of eusociality’ (Rosengren & Pamilo 1983). We developed microsatellite loci from random ampli- There are many polygynous species in the common ant fied polymorphic DNA (RAPD) fragments following the genus Formica that consists of four subgenera (Raptiformica, procedure reported in Hasegawa & Takahashi (2002). The Coptoformica, Serviformica and s. str.). A highly polygynous RAPD reactions were conducted under 45 cycles of 94 °C ant, Formica (s. str.) yessensis, makes huge colonies (so-called for 30 s, 35 °C for 45 s and 72 °C for 2 min. The temperature super colonies) that range over several square kilometres ramp speed was 1.0 °C/s other than 0.3 °C/s between and colony members share all the nests (Higashi 1976). 35 and 72 °C (for detailed conditions, see Hasegawa & New queens mate with nest-mate males on the ground and Takahashi 2002). The products of 40 RAPD primers that return to their own nests (Ito & Imamura 1974). These proved positive for microsatellites (10 repeats of any of AG, habits have attracted researchers to this ant from the CG, TG or AT) were cloned into pUC19 and recombinants viewpoints of both behavioural ecology (e.g. kin selection were subject to a second screening. In total, 20 of 189 and evolution of sociality) and population genetics (e.g. recombinants were positive. Inserts of the positive recom- population viscosity). For both viewpoints, microsatellite binants were amplified independently using the vector’s loci are very useful to examine genetic structure within primers (BcaBESTTM Sequencing Primer RV-M and M13 - 47; a colony and/or a population. Microsatellite primers for TaKaRa) and sequenced using automated sequencers (ABI European F. (s. str.) lugubris (Chapuisat 1996) have been 373S, Applied Biosystems; CEQ2000, Beckman and Coulter). reported to be applicable to European F. (s. str.) truncorum There were 14 different loci with more than five micro- (Sundström 1993, 1995). Although we used the primers satellite repeats in these positive clones. having the provided sequences and followed the polymerase We could design primer pairs for 13 of the 14 loci chain reaction (PCR) procedure in the published papers (Table 1) because the cloned sequence of a locus does not (Sundström 1995; Chapuisat 1996), our tests failed to have a long enough flanking region for primer design. To amplify the target regions of Japanese F. (s. str.) yessensis test the effectiveness of the obtained microsatellite loci, we and F. (s. str.) truncorum (S. Imai, personal observations). checked the amplification of the primer pairs using F. (s. str.) yessensis (20 individuals from 10 nests) and a related Correspondence: Eisuke Hasegawa. Fax: + 81-11-757-5595; E-mail: species, F. (s. str.) truncorum (20 individuals from 10 nests). [email protected] For the loci that were successfully amplified in this test, we

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Table 1 Primers, polymerase chain reaction conditions and core sequence of 13 microsatellites obtained from Formica yessensis

Core sequence MgCl2 Annealing Allele size GenBank Locus name in cloned alleles Primer sequence (5′−3′) (mm) temp. (°C) Cycles (bp) Accession no.

′ ′ Fy2 (CA)19 F: 5 -CGATTAGGGGGATTGGTGTA-3 1.5 43 28 368 AB103291 R: 5′-GGCGTTCGTCAACAGATATG-3′ ′ ′ Fy3 (CT)16 F: 5 -ATTCATATGAGTTACATCGA-3 1.5 45 26 199 AB103292 R: 5′-AGGTAATCAATATATTTAAG-3′ ′ ′ Fy4 (CT)17 F: 5 -TTCCACTGGAGAACCATCGG-3 1.5 45 27 286 AB103293 R: 5′-TGCGTTTGAAATTTCCACAG-3′ ′ ′ Fy5 (CAT)7 F: 5 -ATTTTCACCAAACAATAAGA-3 1.5 48 28 200 AB103294 R: 5′-TATCATACTTGGTCTTACCC-3′ ′ ′ Fy6 (ATT)7(CTT)2 F: 5 -TGTAAAGGTGGGTGTTTTTG-3 1.5 NA NA 205 AB103295 ′ ′ (CGT)13 R: 5 -CGTAACGTATATAAATTAAA-3 ′ ′ Fy7 (CA)16(TA)3 F: 5 -CACACTTATTTACTCTGGCC-3 1.5 53 28 248 AB103296 R: 5′-CAGGCAGAGATAATATTTGC-3′ ′ ′ Fy8 (GT)13AT(GT)5 F: 5 -TGGCGGCCGTTCAAATTTCG-3 1.5 NA NA 200 AB103297 R: 5′-AAAGTTGTTTCTCTCCGACC-3′ ′ ′ Fy9 (CG)4(CA)7 F: 5 -CAAAACACACCTCGTCCACC-3 1.5 48 28 228 AB103298 ′ ′ TCAT(GC)4 R: 5 -GTAACCCGAGCGAGCCCGAA-3 ′ ′ Fy10 (CT)9 F: 5 -ATAAAGTTTTAAATAATCAA-3 1.5 NA NA 150 AB103299 R: 5′-ACTTAGAATCAAATTCGTCC-3′ ′ ′ Fy12 (AG)12 F: 5 -ATGACGATTAGGGGGATTGG-3 1.5 48 28 188 AB103301 R: 5′-TTCTCTTACAACTATTCAAC-3′ ′ ′ Fy13 (CT)16TTT(CT)5 F: 5 -TCGTCGCGGTAAATCATTCC-3 1.5 48 28 204 AB103302 R: 5′-CCCTTTTTCGCCCAATTTCC-3′ ′ ′ Fy14 (AT)8T(AC)11 F: 5 -GAAAGTTTTCATAACACATC-3 1.5 48 28 201 AB103303 ′ ′ (AT)13 R: 5 -AGGATTTATTCCGCGTAATC-3 ′ ′ Fy15 (GA)2G(GA)7 F: 5 -GATCGATAAATTCCAGTGCG-3 1.5 48 28 233 AB103304 ′ ′ A(GA)5 R: 5 -AACCGTTCGAGCCTATCACC-3

NA, not amplified.

checked the degree of polymorphism using four popu- the 13 loci were polymorphic with two to five alleles (see lations of each species (see Table 2). For the cross-genus Table 2). The allele detection method (acrylamide gel elec- amplification test, we used a closely related species from a trophoresis and silver staining) did not allow us to specify different genus, Polyergus samurai, and two males per nest the length of alleles but the observed allele lengths were were genotyped in nine nests for this species. The total distributed around the length of the cloned allele. By DNA of each individual was extracted by boiling the ground sequencing some alleles, we confirmed that the observed tissue of a leg in 300 µL of 5% Chelex resin (Bio-Rad) for 2 h minimum difference of allele length corresponded to a 2- at 55 °C (Walsh et al. 1991). The PCR amplifications were bp difference. carried out in a total volume of 10 µL, which contained 1.5 µL We tested for deviations of genotype frequencies from (c. 10 ng) of template DNA, 2.5 pmol of each microsatellite Hardy–Weinberg equilibrium for each locus in each popu- primer, 400 mm of dNTP, 1 µL of 10 × reaction buffer, lation using χ 2 tests (Table 2; tests were done by hand cal-

1.5 mm MgCl2 and 0.05 U of Taq (TaKaRa). Temperature culations). A significant deviation was observed at a single cycles were as follows; 3 min at 94 °C followed by locus- locus (Fy7) in the Kawayu population in Hokkaido prefec- specific numbers of cycles (see Table 1), i.e. 30 s at 94 °C, ture (χ 2 test, χ 2 = 20.22, P < 0.001 with Bonferroni correc- 30 s at 45–53 °C (for the annealing temperature of each tion for multiple comparisons). The cause of this deviation locus, see Table 1) and 30 s at 72 °C. The PCR products could not be attributed to the existence of null alleles were run on 8% polyacrylamide (mono : bis, 37.5 : 1) gels because there was an excess of heterozygotes in this popu- and visualized by silver staining (Tegelström 1986). A 2-bp lation. We also tested for linkage disequilibrium among difference in length could be discriminated by this method. the polymorphic loci using genepop version 3.3 (Raymond The sequence information of each locus is available from & Rousset 1995). Linkage disequilibrium was detected for databases (GenBank Accession nos AB103291–AB103304). one pair of loci (Fy7 and Fy13, P = 0.017, Fisher’s exact We checked the polymorphism at each locus using four probability test with Bonferroni correction for multiple populations of F. yessensis (four populations, total n = 479) comparisons, cf. Bonferroni corrections were made by and F. truncorum (four populations, total n = 436). Five of hand calculations) in one population (Gotenba in Shizuoka

Table 2 Number of alleles (Na), expected heterozygosity (HE) and observed heterozygosity (HO) at all loci in three species of two different genera

Deviation from Hardy–Weinberg equilibrium

χ2 Locus Species Population nNa HE HO d.f. Significance

Fy3 Formica (s. str.) truncorum Obihiro 99 4 0.286 0.263 9.309 6 NS Kawayu 99 4 0.721 0.556 18.935* 5 NS Moshiri 90 4 0.632 0.622 1.811 6 NS Furano 100 4 0.700 0.571 12.119 6 NS F. (s. str.) yessensis Ishikari 100 4 0.574 0.500 8.970 6 NS Ohnuma 100 3 0.156 0.170 0.863 3 NS Gotenba 100 2 0.180 0.180 0.000 1 NS Norikura 100 1 — — — — — Polyergus samurai Chiba 18 2 0.198 — — — — Fy4 F. (s. str.) truncorum Obihiro 99 2 0.010 0.010 0.003 1 NS Kawayu 99 2 0.030 0.030 0.023 1 NS Moshiri 90 2 0.054 0.056 0.074 1 NS Furano 99 2 0.114 0.101 1.262 1 NS F. (s. str.) yessensis Ishikari 100 2 0.458 0.570 5.987 1 NS Ohnuma 100 2 0.394 0.420 0.428 1 NS Gotenba 95 2 0.172 0.168 0.031 1 NS Norikura 100 2 0.188 0.210 1.376 1 NS P. samurai Chiba 18 1 — — — — — Fy7 F. (s. str.) truncorum Obihiro 93 3 0.515 0.538 5.600* 2 NS Kawayu 96 3 0.520 0.750 21.140 3 P = 0.003 Moshiri 85 4 0.500 0.541 6.226 6 NS Furano 95 3 0.631 0.516 9.124* 2 NS F. (s. str.) yessensis Ishikari 98 3 0.533 0.622 4.682* 1 NS Ohnuma 100 2 0.446 0.490 0.995 1 NS Gotenba 100 2 0.494 0.530 0.534 1 Norikura 99 2 0.448 0.495 1.100 1 NS P. samurai Chiba 18 NA — — — — — Fy13 F. (s. str.) truncorum Obihiro 99 3 0.335 0.354 2.520 3 NS Kawayu 99 4 0.472 0.505 3.050 6 NS Moshiri 89 3 0.269 0.213 5.766 3 NS Furano 100 3 0.411 0.380 0.086* 1 NS F. (s. str.) yessensis Ishikari 100 3 0.289 0.220 2.474* 1 NS Ohnuma 100 3 0.406 0.340 3.241 3 NS Gotenba 100 2 0.086 0.090 0.222 1 NS Norikura 100 1 — — — — — P. samurai Chiba 18 1 — — — — — Fy15 F. (s. str.) truncorum Obihiro — — — — — — — Kawayu — — — — — — — Moshiri 10 3 0.461 0.500 –† — — Furano — — — — – — — F. (s. str.) yessensis Ishikari 8 4 0.690 0.500 –† — — Ohnuma 10 3 0.555 0.600 –† — — Gotenba — — — — — — — Norikura — — — — — — — P. samurai Chiba 18 2 0.278 — — — —

*χ2 values were corrected for combining cells with expected values of < 5. †χ2 test was not done because sample size is too small (n < 10). Deviation from Hardy–Weinberg equilibrium was tested statistically for each locus in each population. NA, not amplified; ND, no data;

NS, not significant; —, calculation is impossible. For P. samurai, HO cannot be calculated because all the examined samples were males, which are haploid in Hymenoptera.

© 2004 Blackwell Publishing Ltd, Molecular Ecology Notes, 4, 200–203 PRIMER NOTE 203 prefecture). In the Gotenba population, one of the four Hasegawa E, Takahashi J (2002) Microsatellite loci for genetic alleles at Fy13 (the shortest allele, frequency 0.045) was research in the hornet Vespa mandarinia and related species. detected only with a specific allele at Fy7 in this population Molecular Ecology Notes, 2, 306–308. Hasegawa E, Tinaut A, Ruano F (2001) Molecular phylogeny of two but this linkage disequilibrium between these two alleles slave-making ants: Rossomyrmex and Polyergus (Hymenoptera: was not observed in the other populations. Formicidae). Annals Zoologici Fennici, 39, 267–271. In P. samurai, although the two loci were monomorphic, Higashi S (1976) Nest proliferation by budding and nest growth two of the five loci were polymorphic (two alleles) and a even types of pleiotropy. International Journal of Developmental primer pair could not amplify the target region (Table 2). Biology, 42, 501–505. The observed alleles at the polymorphic loci corresponded Ito M, Imamura S (1974) Observations on the nuptial flight and in length with the alleles of Formica. A phylogenetic study internidal relationship in a polydomous ant, Formica yessensis Forel. Journal of the Faculty of Science, Hokkaido University, Series showed that P. samurai is the closest sister genus of the VI, 19, 681–694. genus Formica, in which four subgenera are known (Haseg- Raymond M, Rousset F (1995) genepop Version 1.2: population awa et al. 2001). The reported primers are likely to work in genetics software for exact tests and ecumenism. Journal of these Formica subgenera. Thus, we concluded that these Heredity, 86, 248–249. microsatellite markers will be useful in the genetic analysis Rosengren R, Pamilo P (1983) The evolution of polygyny and of Formica ants. polydomy in mound-building Formica ants. Acta Entomolgici Fennici, 42, 65–77. Sundström L (1993) Genetic population structure and sociogenetic Acknowledgements organization in Formica truncorum (Hymenoptera: Formicidae). Behavioral Ecology and Sociobiology, 33, 345–354. We thank Dr A. R. Chittenden for improvement of English usage. Sundström L (1995) Dispersal polymorphism and physiological This study was partly supported by the Grant-in-Aid nos 12640604 condition of males and females in the ant Formica truncorum. and 13440227 (to EH) for Scientific Research from the Ministry of Behavioral Ecology, 6, 132–139. Education, Culture, Sports, Science and Technology, Japan. Tegelström H (1986) Mitochondrial DNA in natural populations: An improved routine for the screening of genetic References variation based on sensitive silver staining. Electrophoresis, 7, 226–229. Chapuisat M (1996) Characterization of microsatellite loci in Formica Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium lugubris B and their variability in other ant species. Molecular for simple extraction of DNA for PCR-based typing from forensic Ecology, 5, 599– 601. material. Biotechniques, 10, 506–513.